Pepper plants with improved pest resistance

ABSTRACT

Pepper plants exhibiting resistance to root knot nematode species are provided, together with methods of producing, identifying, or selecting plants or germplasm with a root knot nematode resistance phenotype. Such plants include pepper plants comprising introgressed genomic regions conferring pest resistance. Compositions, including novel polymorphic markers for detecting plants comprising introgressed pest resistance alleles, are further provided.

REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.16/606,648, filed Oct. 18, 2019, which is a 371 National Stageapplication of International Application No. PCT/US2018/029178, filedApr. 24, 2018, which claims the benefit of U.S. Provisional ApplicationNo. 62/490,554, filed Apr. 26, 2017, which are herein incorporated byreference in their entireties.

INCORPORATION OF SEQUENCE LISTING

A sequence listing containing the file named“SEMB025WO_ST26_Revised.xml” which is 24.3 kilobytes (measured inMS-Windows®) and created on Nov. 14, 2022, and comprises 25 sequences,and is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to the field of plant breeding and morespecifically to methods and compositions for producing pepper plantsexhibiting improved pest resistance.

Background

Pest resistance is an important trait in agriculture, particularly forthe production of food crops. In pepper plants, root knot nematodes(RKN) result in significant yield loss and even plant death. AlthoughRKN resistance alleles have been identified in pepper plants, knownresistance alleles vary in their resistance against the differentspecies of the RKN complex. Deployment of resistance loci starts anevolutionary arms race between pest and breeder leading to newlyemerging species of RKN that are capable of reproducing on pepper plantscarrying previously known forms of RKN resistance. Therefore, it isnecessary that breeders find and develop new sources of RKN resistancewith alternative resistance profiles and genetic variation to maintaingood resistance levels and minimize crop loss in commercial pepperproduction.

SUMMARY

In one aspect, the invention provides a Capsicum annuum plant comprisingan introgressed allele on chromosome 9 that confers increased resistanceto root knot nematodes compared to a plant not comprising said allele,wherein said plant is a non-jalapeno variety, and wherein saidintrogressed allele is located between Marker_6 (SEQ ID NO: 1) andMarker_10 (SEQ ID NO: 21) on chromosome 9. In some embodiments, theplant is a variety selected from the group consisting of Anaheim,Ancho/Poblano, Asian Long Slim, Asian Short, Blocky or Bell, Capia,Cascabel, Cayenne, Chiltepins or Small Hots, Corno di Toro, Cubanelle,‘Fresno Chili’, Hungarian Wax/Banana/Hungarian White, Ornamental,Pasilla, Pimiento, Santa Fe Grande, Serrano, and Waxy peppers. Incertain embodiments, the plant has a blocky type fruit shape, a ¾ longtype fruit shape, or a half long type fruit shape. For example, thefruit of the plant may have a length to width ratio less than 2.5:1,such as a length to width ratio of less than 2:1, or a length to widthratio of between 0.8 to 1.2.

In other embodiments, the Capsicum annuum plant comprises anintrogressed allele flanked in the genome of said plant by position250,338,645 bp and position 250,504,749 bp on chromosome 9 on thephysical map of CM334 v1.55. The introgressed allele may be flanked inthe genome of said plant by marker locus Marker_7 (SEQ ID NO: 6) andmarker locus Marker_9 (SEQ ID NO: 16). In further embodiments, theCapsicum annuum plant comprises an introgressed allele comprising theresistance haplotype of HJA-114-1011. In certain embodiments, theintrogressed allele has been introgressed from PX11435810, a sample ofthe seed of which has been deposited under ATCC Accession No. PTA-13408.In further embodiments, Capsicum annuum plant of the invention areresistant to isolates of the root knot nematode species M. enterolobii,M. javanica, M. arenaria, and M. incognita.

In another aspect, the invention provides a plant part of the Capsicumannuum plant of the invention, for example a cell, a seed, a root, astem, a leaf, a fruit, a flower, or pollen.

In yet a further aspect, a method is provided for producing a Capsicumannuum plant exhibiting resistance to root knot nematodes, comprising:a) crossing a Capsicum annuum plant provided herein with itself or witha second Capsicum annuum plant of a different genotype to produce one ormore progeny plants; and b) selecting a progeny plant comprising saidintrogressed allele. In certain embodiments, selecting said progenyplant comprises identifying a genetic marker genetically linked to saidintrogressed allele. In some embodiments, selecting said progeny plantcomprises identifying a genetic marker within or genetically linked to agenomic region between 250,338,645 bp and 250,504,749 bp on chromosome 9on the physical map of CM334 v1.55. Selecting said progeny may furthercomprise identifying a genetic marker within or genetically linked to agenomic region flanked in the genome of said plant by marker locusMarker_7 (SEQ ID NO: 6) and marker locus Marker_9 (SEQ ID NO: 16), forexample detecting at least one polymorphism at a locus selected from thegroup consisting of marker locus Marker_7 (SEQ ID NO: 6), marker locusMarker_8 (SEQ ID NO: 11), and marker locus Marker_9 (SEQ ID NO: 16). Theprogeny plant may be an F₂-F₆ progeny plant, and the methods providedmay comprises backcrossing, for example from 2-7 generations ofbackcrossing.

In another aspect, a method is provided for producing a Capsicum annuumplant exhibiting resistance to root knot nematodes, comprisingintrogressing into a plant a root knot nematode resistance allele,wherein said resistance allele is defined as located in a genomic regionbetween 250,338,645 bp and 250,504,749 bp on chromosome 9 on thephysical map of CM334 v1.55. In certain embodiments, said genomic regionis flanked by Marker_7 (SEQ ID NO: 6) and marker locus Marker_9 (SEQ IDNO: 16). The method may comprise backcrossing, marker-assistedselection, or assaying for root knot nematode resistance.

In a further aspect, Capsicum annuum plant is provided that isobtainable by a method comprising the steps of: a) crossing a Capsicumannuum plant provided herein with itself or with a second Capsicumannuum plant of a different genotype to produce one or more progenyplants; and b) selecting a progeny plant comprising said introgressedallele. Selecting said progeny plant may comprise identifying a geneticmarker genetically linked to said introgressed allele, for exampleidentifying a genetic marker within or genetically linked to a genomicregion flanked in the genome of said plant by marker locus Marker_7 (SEQID NO: 6) and marker locus Marker_9 (SEQ ID NO: 16). Selecting a progenyplant may further comprise detecting at least one polymorphism at alocus selected from the group consisting of marker locus Marker_7 (SEQID NO: 6), marker locus Marker_8 (SEQ ID NO: 11), and marker locusMarker_9 (SEQ ID NO: 16). In some embodiments, said progeny plant is anF₂-F₆ progeny plant. Producing said progeny plant may comprisebackcrossing, for example from 2-7 generations of backcrossing.

In yet a further aspect, Capsicum annuum plant is provided that isobtainable by a method comprising the step of: introgressing into aplant a root knot nematode resistance allele, wherein said resistanceallele is defined as located in a genomic region between 250,338,645 bpand 250,504,749 bp on chromosome 9 on the physical map of CM334 v1.55.In some embodiments, said genomic region is flanked by Marker_7 (SEQ IDNO: 6) and marker locus Marker_9 (SEQ ID NO: 16). In furtherembodiments, introgressing comprises backcrossing, marker-assistedselection, or assaying for said root knot nematode resistance.

In another aspect, methods are provided of selecting a Capsicum annuumplant exhibiting resistance to root knot nematodes, comprising: a)crossing a Capsicum annuum plant provided herein with itself or with asecond Capsicum annuum plant of a different genotype to produce one ormore progeny plants; and b) selecting a progeny plant comprising saidintrogressed allele. In certain embodiments, selecting said progenyplant comprises identifying a genetic marker genetically linked to saidintrogressed allele. Selecting said progeny plant may also compriseidentifying a genetic marker within or genetically linked to a genomicregion between 250,338,645 bp and 250,504,749 bp on chromosome 9 on thephysical map of CM334 v1.55. In additional embodiments, said progenyplant comprises identifying a genetic marker within or geneticallylinked to a genomic region flanked in the genome of said plant by markerlocus Marker_7 (SEQ ID NO: 6) and marker locus Marker_9 (SEQ ID NO: 16).Selecting a progeny plant may also comprise detecting at least onepolymorphism at a locus selected from the group consisting of markerlocus Marker_7 (SEQ ID NO: 6), marker locus Marker_8 (SEQ ID NO: 11),and marker locus Marker_9 (SEQ ID NO: 16). Said progeny plant may be anF₂-F₆ progeny plant. In some embodiments, producing said progeny plantcomprises backcrossing, for example from 2-7 generations ofbackcrossing.

In a further aspect, methods are provided of producing a Capsicum annuumplant exhibiting resistance to root knot nematodes, comprisingintrogressing into a plant a root knot nematode resistance allele,wherein said resistance allele is defined as located in a genomic regionbetween 250,338,645 bp and 250,504,749 bp on chromosome 9 on thephysical map of CM334 v1.55. In certain embodiments, said genomic regionis flanked by Marker_7 (SEQ ID NO: 6) and marker locus Marker_9 (SEQ IDNO: 16). Introgressing may comprise backcrossing, marker-assistedselection, or assaying for said root knot nematode resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Fruit of jalapeno peppers are characterized by a typical bulletshape with a 2.5:1 length to width ratio. Fruit is generally harvestedat the immature green stage.

FIG. 2 : Fruit of blocky peppers are generally characterized by a lengthto width ratio of 0.8 to 1.2, while in some regions this ratio can bebetween 0.8 and 1.4. Colors of the fruit can vary. The most commoncolors are white, purple, and green at the immature stage, and red,yellow, green, orange, and brown at the mature fruit stage

FIG. 3 : Genotype analysis of the nematode resistance region onchromosome 9.

FIG. 4 : Fine mapping of the RKN resistance locus on chromosome 9. Agenotype of “A” refers to a homozygous allele from HJA-114-1011(Resistant parent) while “B” is homozygous for HJA-114-1096 (Sensitiveparent). The Haplotype LSMeans represent the mean gallcount for eachhaplotype. A lower number indicates better resistance against M.enterolobii.

FIG. 5 : Genetic map of the nematode resistance region on chromosome 5showing marker positions and the position of the N-locus, Mel, and theHJA-114-1011 locus.

DETAILED DESCRIPTION

Root knot nematodes (RKN) are microscopic roundworms of the genusMeloidogyne that parasitize the roots of a wide variety of crop plants,resulting in yield loss from infected fields. Several sources of RKNresistance are known in the field of pepper breeding. However, whilethese genes are known to provide some resistance to isolates of M.javanica, M. incognita or M. arenaria, the resistance against M.enterolobii for these sources is either unknown or not sufficientlyrobust. As resistance against M. enterolobii is becoming more importantdue to its aggressiveness and rapid emergence as a primary pest inimportant pepper growing regions of the world, such as Mexico and theUS, new broad spectrum RKN resistance loci are needed.

The invention therefore provides novel RKN resistance loci on chromosome9 of the Capsicum annuum genome that provides resistance to M. icognita,M. arenaria and M. javanica and to the emerging M. enterolobii species.Resistance genes that confer resistance to this emerging nematodespecies have not been previously described. Surprisingly, although thenovel RKN resistance locus of the invention is located among knownnematode resistance loci in pepper on chromosome 9, fine mapping of thisnovel locus demonstrates that it is distinct from and geneticallyunrelated to known sources of RKN resistance.

The invention additionally provides pepper plants comprising novelintrogressions from donor lines that confer RKN resistance.Specifically, plants comprising the introgressed alleles describedherein provide broad spectrum resistance to M. icognita, M. arenaria, M.javanica, and M. enterolobii. The invention therefore provides achromosomal segment of approximately 4.8 cM located betweenapproximately 109.87 cM and about 114.68 cM on the Monsanto linkage mapof chromosome 9 that confers broad-spectrum resistance to RKN in pepperplants.

The invention further provides novel trait-linked markers that allow theaccurate identification and tracking of the aforementioned chromosomalregions during plant breeding. In particular embodiments, the inventionprovides genetic markers within or genetically linked to a genomicregion between about 109.87 cM and about 114.68 cM on chromosome 9.Other embodiments provide novel markers Marker _6 (SEQ ID NO: 1)comprising a SNP [C/A], Marker _7 (SEQ ID NO:6) comprising a SNP [T/A]at 250,504,749 bp of the public genome of CM334 v1.55, Marker _8 (SEQ IDNO:11) comprising a SNP [A/G] at 250,338,645bp of the public genomeCM334 v1.55, Marker_9 (SEQ ID NO:16) comprising a SNP [T/C] at250,502,864bp of the public genome CM334 v1.55, and Marker_10 (SEQ IDNO: 21) comprising a SNP [G/C] at 241,570,991bp of the public genomeCM334 v1.55, which are useful in detection and tracking of plantscomprising RKN resistance. The official public genome of CM334 ispublically available (Kim, et al. Nature Genetics 46, 270-278 (2014)),and can be accessed through the SOL genomics network.

I. Genomic Regions, Alleles, and Polymorphisms Associated with RKNResistance in Pepper Plants

RKN are soil-borne pests that infect plant roots, causing severedecreases in yield or even plant death. A broad range of crop plants issusceptible to RKN infection, leading to significant yield lossworldwide. Cultivar resistance is the most economically feasible way ofcontrolling RKN infection due the high cost and variable performance ofpesticide treatments. Intensive efforts have therefore been made toidentify effective sources of RKN resistance. However, previously knownintrogressions from RKN resistant accessions confer resistance to only asubset of RKN species.

RKN species include, but are not limited to, Meloidogyne acronea,Meloidogyne arenaria, Meloidogyne artiellia, Meloidogyne brevicauda,Meloidogyne chitwoodi, Meloidogyne coffeicola, Meloidogyne exigua,Meloidogyne fruglia, Meloidogyne gajuscus, Meloidogyne hapla,Meloidogyne incognita, Meloidogyne javanica, Meloidogyne enterolobii(also known as Meloidogyne mayaguensis), Meloidogyne naasi, Meloidogynepartityla, and Meloidogyne thamesi. The M. icognita, M. arenaria and M.javanica and M. enterolobii species are of particular relevance as peststo commercial pepper plants.

Several sources of RKN resistance are known in the field of pepperbreeding including CM334, PI322719 (also known as PM687), PI201234 (alsoknown as PM217), and Carolina Cayenne. While the resistance profilesdiffer between these sources, previously known resistance genes havebeen mapped to a gene cluster at one end of chromosome 9. Of the knownresistance genes, Mel from PI201234, Me3 from PI322719 (likely the sameas Mel from CM334), and the N-gene from Carolina Cayenne are currentlywidely used in breeding programs. However, while these genes are knownto provide some resistance to isolates of M. javanica, M. incognita orM. arenaria, the resistance against M. enterolobii is insufficient forthose genes tested in the public. The newly identified resistance locusfrom HJA-114-1011 described herein provides resistance to M.enterolobii, M. incognita, M. javanica, and M. arenaria.

In certain embodiments, the invention therefore provides pepper plantscomprising donor DNA from a RKN resistant line that maps betweenapproximately 109.87 cM and about 114.68 cM on chromosome 9. This novelintrogression provides resistance to RKN species including M.enterolobii. Genomic regions as described herein can be obtained fromany wild or cultivated plant or line, including in certain embodimentspepper line PX11435810 (a sample of the seed of which has been depositedwith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110-2209 USA under ATCC Accession No.PTA-13408, Dec. 12, 2012), which is a jalapeno hybrid havingHJA-114-1011 as a parent and comprising the genomic regions as describedherein. Plants provided herein include plants comprising the geneticsource for the RKN resistance trait from pepper line PX11435810.

Other embodiments of the invention provide novel markers for theselection of the RKN resistance locus on chromosome 9 described herein.Examples of such markers include Marker_7, Marker_8, and Marker_9, whichhave been shown to be genetically linked to RKN resistance in plants.

II. Introgression of Genomic Regions Associated with RKN Resistance

Marker-assisted introgression involves the transfer of a chromosomalregion defined by one or more markers from a first genetic background toa second. Offspring of a cross that contain the introgressed genomicregion can be identified by the combination of markers characteristic ofthe desired introgressed genomic region from a first genetic backgroundand both linked and unlinked markers characteristic of the secondgenetic background.

The present invention provides novel markers for identifying andtracking introgression of one or more of the genomic regions disclosedherein from an RKN resistant plant into a cultivated line. The inventionfurther provides markers for identifying and tracking the novelintrogressions disclosed herein during plant breeding, such as markersMarker_7, Marker_8, and Marker_9.

Markers within or linked to any of the genomic intervals of the presentinvention may be useful in a variety of breeding efforts that includeintrogression of genomic regions associated with pest resistance into adesired genetic background. For example, a marker within 40 cM, 20 cM,15 cM, 10 cM, 5cM, 2 cM, or 1 cM of a marker associated with pestresistance described herein can be used for marker-assistedintrogression of genomic regions associated with a pest resistancephenotype.

Pepper plants comprising one or more introgressed regions associatedwith a desired phenotype wherein at least 10%, 25%, 50%, 75%, 90%, or99% of the remaining genomic sequences carry markers whose alleles matchthe recurrent parent genotype outside of the region targeted for pestresistance introgression are also provided. Pepper plants comprising anintrogressed region closely linked to, or adjacent to, the genomicregions and markers provided herein and associated with a pestresistance phenotype are also provided.

III. Development of RKN Resistant Pepper Varieties

For most breeding objectives, commercial breeders work within germplasmthat is “cultivated,” “cultivated type,” or “elite.” As used herein,“elite” or “cultivated” variety means a variety that has resulted frombreeding and selection for superior horticultural performance for use inagriculture. This germplasm is easier to breed because it generallyperforms well when evaluated for horticultural performance. A number ofcultivated pepper types have been developed, which are agronomicallyelite and appropriate for commercial cultivation. However, theperformance advantage a cultivated germplasm provides can be offset by alack of allelic diversity. Breeders generally accept this tradeoffbecause progress is faster when working with cultivated material thanwhen breeding with genetically diverse sources.

In contrast, when cultivated germplasm is crossed with non-cultivatedgermplasm, a breeder can gain access to novel alleles from thenon-cultivated type. However, this approach generally presentssignificant difficulties due to fertility problems associated withcrosses between diverse lines, and negative linkage drag from thenon-cultivated parent. For example, non-cultivated pepper lines canprovide alleles associated with pest resistance. However, thesenon-cultivated lines may have poor horticultural qualities such asundesirable fruit shape, undesirable immature fruit color, small fruitsize, or low yield.

The process of introgres sing desirable resistance genes fromnon-cultivated lines into elite cultivated lines while avoiding problemswith linkage drag or low heritability of the desired trait in crosseswith the non-cultivated lines is a long and often arduous process.Success in deploying alleles derived from wild relatives thereforestrongly depends on minimal or truncated introgressions that lackdetrimental effects and reliable marker assays that replace phenotypicscreens. Success is further defined by simplifying genetics for keyattributes to allow focus on genetic gain for quantitative traits suchas pest resistance. Moreover, the process of introgressing genomicregions from non-cultivated lines can be greatly facilitated by theavailability of accurate markers for marker-assisted selection (MAS).

One of skill in the art would therefore understand that the alleles,polymorphisms, and markers provided by the invention allow the trackingand introduction of any of the genomic regions identified herein intoany genetic background. In addition, the genomic regions associated withpest resistance disclosed herein can be introgressed from one genotypeto another and tracked using MAS. Thus, the novel, accurate markersassociated with pest resistance provided herein will facilitate thedevelopment of pepper plants having beneficial phenotypes. For example,seed can be genotyped using the markers of the present invention inorder to select for plants comprising desired genomic regions associatedwith pest resistance, without the need for growing plants to maturity toevaluate the phenotype. Moreover, MAS allows identification of plantshomozygous or heterozygous for a desired introgression.

Phenotypic evaluation of large plant populations is time-consuming,resource-intensive and not reproducible in every environment.Marker-assisted selection offers a feasible alternative. Molecularassays designed to detect unique polymorphisms, such as SNPs, areversatile. However, they may fail to discriminate alleles within andamong pepper species in a single assay. Structural rearrangements ofchromosomes such as deletions impair hybridization and extension ofsynthetically labeled oligonucleotides. In the case of duplicationevents, multiple copies are amplified in a single reaction withoutdistinction. The development and validation of accurate and highlypredictive markers are therefore essential for successful MAS breedingprograms.

In certain embodiments, the genomic regions identified herein may beintrogressed from any Capsicum annuum type into any Capsicum annuumtype. Types of Capsicum annuum include, but are not limited to Anaheim,Ancho/Poblano, Asian long slim, Asian short, Blocky or Bell, Capia,Cascabel, Cayenne, Chiltepins or Small Hots, Corno di Toro, Cubanelle,‘Fresno Chili’, Jalapeno, Ornamental, Pasilla, Pimiento, Santa FeGrande, Serrano, and Waxy peppers, including Hungarianwax/Banana/Hungarian white.

In certain embodiments, the identified genomic regions are introducedfrom jalapeno type pepper into a non-jalapeno variety. Pepper fruitshapes are well-known to those skilled in the art of pepper breeding.Jalapeno peppers are a type of Capsicum annuum that have acharacteristic fruit shape. Jalapeno fruits are typically bullet-shapedand have a length to width ratio of about 2.5 to 1 (FIG. 1 ). Forexample, a fruit having a length of about 10 cm would be expected to beabout 4 cm wide. The fruit typically has thick walls of about 5-6 mm andthe dry matter content is normally around 7%. The fruit of most plantsdevelops from a medium green at the immature stage to red at the maturestage. As a commercial product, the fruits are harvested at the greenstage. The pungency of jalapeno peppers varies from 0 units to over 5000units on the Scoville scale.

Non-jalapeno varieties include blocky type peppers, half long-typepeppers, and ¾ long-type peppers. In certain examples, non-jalapenovarieties include, but are not limited to Anaheim, Ancho/Poblano, AsianLong Slim, Asian Short, Blocky or Bell, Capia, Cascabel, Cayenne,Chiltepins or Small Hots, Corno di Toro, Cubanelle, ‘Fresno Chili’,Hungarian Wax/Banana/Hungarian White, Ornamental, Pasilla, Pimiento,Santa Fe Grande, Serrano, and Waxy peppers. Non-jalapeno varietiesproduce fruit with a length to width ratio of less than about 2.0 to 1,for example less than about 1.2. Non-jalapeno varieties include pepperplants producing fruit with a length to width ratio from 0.8 to 1.2. Asused herein, blocky type pepper refers to a pepper wherein the length ofthe fruit is about the same as the width of the fruit. For example, thelength of the fruit is about 0.8, about 0.9, about 1.0, about 1.1, orless than 1.2 of the width of the fruit (FIG. 2 ). As used herein, halflong type pepper refers to a pepper wherein the length of the fruit isabout 1.2 to about 1.5 of the width of the fruit. As used herein, ¾ longtype pepper, often known as lamuyo refers to a pepper wherein the lengthof the fruit is more than about 1.5 of the width of the fruit. Thesepeppers can have a variety of different colors, for example white,purple, and green at the immature stage, and for example red, yellow,green, orange, and brown at the mature fruit stage. It is alsowell-known that definitions vary regionally. For example, in the UnitedStates peppers with a length to with ratio of 1.2 to 1.4 are oftenreferred to as “deep blocky”, while the terms half long and lamuyo areused interchangeably for sweet peppers with a length to width ratio over1.4.

Many desirable traits that are successfully introduced throughintrogression can also be introduced directly into a plant by the use ofmolecular techniques. One aspect of the invention includes plants with agenome that has been changed by any method using site-specific genomemodification techniques. Techniques of site-specific genome modificationinclude the use of enzymes such as, endonucleases, recombinases,transposases, helicases and any combination thereof. In one aspect, anendonuclease is selected from a meganuclease, a zinc-finger nuclease(ZFN), a transcription activator-like effector nucleases (TALEN), anArgonaute, and an RNA-guided nuclease, such as a CRISPR associatednuclease.

In another aspect, the endonuclease is a dCas9-recombinase fusionprotein. As used herein, a “dCas9” refers to a Cas9 endonuclease proteinwith one or more amino acid mutations that result in a Cas9 proteinwithout endonuclease activity, but retaining RNA-guided site-specificDNA binding. As used herein, a “dCas9-recombinase fusion protein” is adCas9 with a protein fused to the dCas9 in such a manner that therecombinase is catalytically active on the DNA.

Non-limiting examples of recombinase include a tyrosine recombinaseattached to a DNA recognition motif provided herein is selected from thegroup consisting of a Cre recombinase, a Gin recombinase a Flprecombinase, and a Tnp 1 recombinase. In an aspect, a Cre recombinase ora Gin recombinase provided herein is tethered to a zinc-fingerDNA-binding domain, or a TALE DNA-binding domain, or a Cas9 nuclease. Inanother aspect, a serine recombinase attached to a DNA recognition motifprovided herein is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In another aspect, aDNA transposase attached to a DNA binding domain provided herein isselected from the group consisting of a TALE-piggyBac and TALE-Mutator.

Site-specific genome modification enzymes, induce a genome modificationsuch as a double-stranded DNA break (DSB) or single-strand DNA break atthe target site of a genomic sequence that is then repaired by thenatural processes of homologous recombination (HR) or non-homologousend-joining (NHEJ). Sequence modifications then occur at the cleavedsites, which can include deletions or insertions that result in genedisruption in the case of NHEJ, or integration of exogenous sequences byhomologous recombination.

Another aspect of the invention includes transgenic plant cells,transgenic plant tissues, transgenic plants, and transgenic seeds thatcomprise the recombinant DNA molecules and engineered proteins providedby the invention. These cells, tissues, plants, and seeds comprising therecombinant DNA molecules and engineered proteins exhibit resistance toRKN. Suitable methods for transformation of host plant cells for usewith the current invention include virtually any method by which DNA canbe introduced into a cell (for example, where a recombinant DNAconstruct is stably integrated into a plant chromosome) and are wellknown in the art. An exemplary and widely utilized method forintroducing a recombinant DNA construct into plants is the Agrobacteriumtransformation system, which is well known to those of skill in the art.Another exemplary method for introducing a recombinant DNA constructinto plants is insertion of a recombinant DNA construct into a plantgenome at a pre-determined site by methods of site-directed integration.Transgenic plants can be regenerated from a transformed plant cell bythe methods of plant cell culture. A transgenic plant homozygous withrespect to a transgene (that is, two allelic copies of the transgene)can be obtained by self-pollinating (selfing) a transgenic plant thatcontains a single transgene allele with itself, for example an RO plant,to produce R1 seed. One fourth of the R1 seed produced will behomozygous with respect to the transgene. Plants grown from germinatingR1 seed can be tested for zygosity, using a SNP assay, DNA sequencing,or a thermal amplification assay that allows for the distinction betweenheterozygotes and homozygotes, referred to as a zygosity assay.

IV. Molecular Assisted Breeding Techniques

Genetic markers that can be used in the practice of the presentinvention include, but are not limited to, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),simple sequence repeats (SSRs), simple sequence length polymorphisms(SSLPs), single nucleotide polymorphisms (SNPs), insertion/deletionpolymorphisms (Indels), variable number tandem repeats (VNTRs), andrandom amplified polymorphic DNA (RAPD), isozymes, and other markersknown to those skilled in the art. Marker discovery and development incrop plants provides the initial framework for applications tomarker-assisted breeding activities (U.S. Patent Pub. Nos.:2005/0204780, 2005/0216545, 2005/0218305, and 2006/00504538). Theresulting “genetic map” is the representation of the relative positionof characterized loci (polymorphic nucleic acid markers or any otherlocus for which alleles can be identified) to each other.

Polymorphisms comprising as little as a single nucleotide change can beassayed in a number of ways. For example, detection can be made byelectrophoretic techniques including a single strand conformationalpolymorphism (Orita et al. (1989) Genomics, 8(2), 271-278), denaturinggradient gel electrophoresis (Myers (1985) EPO 0273085), or cleavagefragment length polymorphisms (Life Technologies, Inc., Gathersberg,MD), but the widespread availability of DNA sequencing often makes iteasier to simply sequence amplified products directly. Once thepolymorphic sequence difference is known, rapid assays can be designedfor progeny testing, typically involving some version of PCRamplification of specific alleles (PASA; Sommer, et al. (1992)Biotechniques 12(1), 82-87), or PCR amplification of multiple specificalleles (PAMSA; Dutton and Sommer (1991) Biotechniques, 11(6),700-7002).

Polymorphic markers serve as useful tools for assaying plants fordetermining the degree of identity of lines or varieties (U.S. Pat. No.6,207,367). These markers form the basis for determining associationswith phenotypes and can be used to drive genetic gain. In certainembodiments of methods of the invention, polymorphic nucleic acids canbe used to detect in a pepper plant a genotype associated with pestresistance, identify a pepper plant with a genotype associated with pestresistance, and to select a pepper plant with a genotype associated withpest resistance. In certain embodiments of methods of the invention,polymorphic nucleic acids can be used to produce a pepper plant thatcomprises in its genome an introgressed locus associated with pestresistance. In certain embodiments of the invention, polymorphic nucleicacids can be used to breed progeny pepper plants comprising a locus orloci associated with pest resistance.

Genetic markers may include “dominant” or “codominant” markers.“Codominant” markers reveal the presence of two or more alleles (two perdiploid individual). “Dominant” markers reveal the presence of only asingle allele. Markers are preferably inherited in codominant fashion sothat the presence of both alleles at a diploid locus, or multiplealleles in triploid or tetraploid loci, are readily detectable, and theyare free of environmental variation, i.e., their heritability is 1. Amarker genotype typically comprises two marker alleles at each locus ina diploid organism. The marker allelic composition of each locus can beeither homozygous or heterozygous. Homozygosity is a condition whereboth alleles at a locus are characterized by the same nucleotidesequence. Heterozygosity refers to different conditions of the allele ata locus.

Nucleic acid-based analyses for determining the presence or absence ofthe genetic polymorphism (i.e. for genotyping) can be used in breedingprograms for identification, selection, introgression, and the like. Awide variety of genetic markers for the analysis of geneticpolymorphisms are available and known to those of skill in the art. Theanalysis may be used to select for genes, portions of genes, QTL,alleles, or genomic regions that comprise or are linked to a geneticmarker that is linked to or associated with pest resistance in pepperplants.

As used herein, nucleic acid analysis methods include, but are notlimited to, PCR-based detection methods (for example, TaqMan assays),microarray methods, mass spectrometry-based methods and/or nucleic acidsequencing methods, including whole genome sequencing. In certainembodiments, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

One method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al. 1986 Cold Spring Harbor Symp. Quant. Biol.51:263-273; European Patent 50,424; European Patent 84,796; EuropeanPatent 258,017; European Patent 237,362; European Patent 201,184; U.S.Pat. Nos. 4,683,202; 4,582,788; and 4,683,194), using primer pairs thatare capable of hybridizing to the proximal sequences that define apolymorphism in its double-stranded form. Methods for typing DNA basedon mass spectrometry can also be used. Such methods are disclosed inU.S. Pat. Nos. 6,613,509 and 6,503,710, and references found therein.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039;7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of whichare incorporated herein by reference in their entirety. However, thecompositions and methods of the present invention can be used inconjunction with any polymorphism typing method to type polymorphisms ingenomic DNA samples. These genomic DNA samples used include but are notlimited to, genomic DNA isolated directly from a plant, cloned genomicDNA, or amplified genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods, for example as disclosed in U.S. Pat. No. 5,800,944 wheresequence of interest is amplified and hybridized to probes followed byligation to detect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al.,Bioinformatics 21:3852-3858 (2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening ofa plurality of polymorphisms. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283.

In another method for detecting polymorphisms, SNPs and Indels can bedetected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe having a 5′ fluorescentreporter dye and a 3′ quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR, forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle DNA polymerase with 5′→3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

In another embodiment, a locus or loci of interest can be directlysequenced using nucleic acid sequencing technologies. Methods fornucleic acid sequencing are known in the art and include technologiesprovided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience(Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-CORBiosciences (Lincoln, Nebr.), NimbleGen Systems (Madison, Wis.),Illumina (San Diego, Calif.), and VisiGen Biotechnologies (Houston,Tex.). Such nucleic acid sequencing technologies comprise formats suchas parallel bead arrays, sequencing by ligation, capillaryelectrophoresis, electronic microchips, “biochips,” microarrays,parallel microchips, and single-molecule arrays.

V. Definitions

The following definitions are provided to better define the presentinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which pepper plants canbe regenerated, plant calli, plant clumps and plant cells that areintact in plants or parts of plants such as pollen, flowers, seeds,leaves, stems, and the like.

As used herein, the term “population” means a genetically heterogeneouscollection of plants that share a common parental derivation.

As used herein, the terms “variety” and “cultivar” mean a group ofsimilar plants that by their genetic pedigrees and performance can beidentified from other varieties within the same species.

As used herein, an “allele” refers to one of two or more alternativeforms of a genomic sequence at a given locus on a chromosome.

A “quantitative trait locus” (QTL) is a chromosomal location thatencodes for at least a first allele that affects the expressivity of aphenotype.

As used herein, a “marker” means a detectable characteristic that can beused to discriminate between organisms. Examples of such characteristicsinclude, but are not limited to, genetic markers, biochemical markers,metabolites, morphological characteristics, and agronomiccharacteristics.

As used herein, the term “phenotype” means the detectablecharacteristics of a cell or organism that can be influenced by geneexpression.

As used herein, the term “genotype” means the specific allelic makeup ofa plant.

As used herein, “elite” or “cultivated” variety means any variety thathas resulted from breeding and selection for superior agronomicperformance. An “elite plant” refers to a plant belonging to an elitevariety. Numerous elite varieties are available and known to those ofskill in the art of pepper breeding. An “elite population” is anassortment of elite individuals or varieties that can be used torepresent the state of the art in terms of agronomically superiorgenotypes of a given crop species, such as pepper. Similarly, an “elitegermplasm” or elite strain of germplasm is an agronomically superiorgermplasm.

As used herein, the term “introgressed” or “introgression,” when used inreference to a genetic locus, refers to a genetic locus that has beenintroduced into a new genetic background, such as through backcrossing.Introgression of a genetic locus can be achieved through plant breedingmethods and/or by molecular genetic methods. Such molecular geneticmethods include, but are not limited to, various plant transformationtechniques and/or methods that provide for homologous recombination,non-homologous recombination, site-specific recombination, and/orgenomic modifications that provide for locus substitution or locusconversion.

As used herein, the term “linked” or “genetically linked,” when used inthe context of nucleic acid markers and/or genomic regions, means thatthe markers and/or genomic regions are located in proximity on the samelinkage group or chromosome such that they tend to segregate together atmeiosis.

As used herein, “resistance locus” means a locus associated withresistance or tolerance to a pest. For instance, a resistance locusaccording to the present invention may, in one embodiment, controlresistance or susceptibility to RKN.

As used herein, “resistance allele” means the nucleic acid sequenceassociated with resistance or tolerance to a pest.

As used herein, “resistance” or “improved resistance” in a plant topests is an indication that the plant is more able to reduce pest damagethan a non-resistant or less-resistant plant. One of skill willappreciate that plant resistance to pests varies widely, and canrepresent a spectrum of more-resistant or less-resistant phenotypes.However, by simple observation, one of skill can generally determine therelative resistance of different plants, plant varieties, or plantfamilies to pests, and furthermore, will also recognize the phenotypicgradations of “resistance.”

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more,”unless specifically noted. The terms “comprise,” “have” and “include”are open-ended linking verbs. Any forms or tenses of one or more ofthese verbs, such as “comprises,” “comprising,” “has,” “having,”“includes” and “including,” are also open-ended. For example, any methodthat “comprises,” “has” or “includes” one or more steps is not limitedto possessing only those one or more steps and also covers otherunlisted steps. Similarly, any plant that “comprises,” “has” or“includes” one or more traits is not limited to possessing only thoseone or more traits and covers other unlisted traits.

EXAMPLES Example 1 Identification of a Resistance Source

A jalapeno pepper inbred line, HJA-114-1011, was identified in a screenas a source of resistance to M. enterolobii, M. incognita, M. javanica,and M. arenaria. HJA-114-1011 is a plant selection from the openpollinated variety ‘Ole’.

Example 2 Genotyping of Resistant Plants

To determine if the resistance of HJA-114-1011 is distinct from any ofthe known resistance sources, a genetic similarity analysis wasperformed. SNP markers in the nematode resistance region of chromosome 9were identified and used to determine the genotype of HJA-114-1011.Clustering analysis based on genetic similarity was used to compare thegenotype of HJA-114-1011 to several inbred lines and resistance sourceswith known resistance genes (FIG. 3 ). It was found that all linescomprising HJA-114-1011 resistance were clustered in a distinct groupfrom lines comprising resistance from other sources. This analysisindicated that the haplotype shared by HJA-114-1011 and its derivativesin this genomic region is highly unrelated (genetic similarity of 50%)to haplotypes associated with Mel and N-gene sources. No sharedhaplotype could be identified between HJA-114-1011 and the Mel donorwithin the nematode gene cluster on chromosome 9. This suggests that RKNresistance from HJA-144-1011 is not related to Mel. In fact, eachresistance gene formed its own cluster in the phylogenetic tree and isthus highly genetically distinct from the other resistance genes. Thisdemonstrates that the HJA-114-1011 nematode resistance locus is uniqueand unrelated to the N-gene, Mel, and Mel resistance loci.

Example 3 QTL Mapping

A recombinant inbred line (RIL) population from a cross betweenHJA-114-1096 (nematode sensitive) and HJA-114-1011 (nematode resistant)was utilized to map the QTL locus conferring resistance to M.enterolobii from HJA-114-1011. 150 individuals from this RIL populationwere screened in a controlled bioassay using a pure M. enterolobiiisolate. The bioassay consists of the tester lines, positive andnegative controls. The resistant parent (HJA-114-1011) was used as apositive control, and the sensitive parent (HJA-114-1096) was used as anegative control. Other negative controls were, for example, CarolinaCayenne, Spartacus or GCW. Two-week-old seedlings were inoculated with500 nematodes per plant by pipetting a 1 mL of a homogeneous nematodesuspension (500 nematodes/mL) into an approximately 3 cm deep hole atthe base of the plant. Preferably fresh inoculum is used, but inoculumthat has been stored at 4° C. for one day can also be used in thisbioassay. Plants were subsequently maintained between 18° C. and 25° C.,with the optimal temperature of 22° C. Excessive watering was avoidedfor the first 3 days to ensure that the nematodes would not wash away.The bioassay screen had 3 replicates of 10 plants per line and all gallswere counted for each plant six weeks post inoculation. These countswere used to calculate the LS mean for each line, which was used in thesubsequent QTL analysis.

The same RIL individuals were genotyped using TaqMan markers spacedacross the genome which were polymorphic between the parental lines. Thecombination of genome wide genotypes and gall count phenotypes were usedto QTL map nematode resistance coming from HJA-114-1011. One majoreffect QTL was found on chromosome 9 located between 100-122 cM andexplains 64.4% of the phenotypic variation. The 2-LOD interval waspositioned at 110.3-113.7 cM, while the 1-LOD interval was similar andlocalized at 110.4-113 cM.

To fine map the chromosome 9 QTL, haplotypes of the 100-122 cM region onchromosome 9 were identified within the RIL population (FIG. 4 ).Fourteen different haplotypes were found with varying numbers of RILindividuals contributing to each haplotype. The LS means for gall countwere calculated across each haplotype group (FIG. 4 ). Based on thisanalysis the region conferring resistance from HJA-114-1011 wasdetermined to be between 109-114 cM, which approximately correlates to110-112 cM on the consensus map. Marker_7, Marker_8, and Marker _9 canthus be used to select for the resistance locus of HJA-114-1011 (Table1). Adding this region to the genetic map of chromosome 9 region, showsthat the HJA-114-1011 region does not overlap with well-known resistanceloci N and Mel (FIG. 5 ).

TABLE 1 Markers associated with the resistance locus of HJA-114-1011.Marker Forward Reverse Public SNP Sequence Primer Primer Probe 1 Probe 2position (SEQ SNP (SEQ (SEQ (SEQ (SEQ CM334 v.1.55 ID NO.) PositionAllele ID NO.) ID NO.) ID NO.) ID NO.) Marker_6 1 111 C/A 2 3 4 5Marker_7 250,504,749 6 130 T/A 7 8 9 10 Marker_8 25,0338,645 11 151 A/G12 13 14 15 Marker_9 250,502,864 16 151 T/C 17 18 19 20 Marker_10241570991 21 151 G/C 22 23 24 25

Example 4 Sequence Analysis

Sequence analysis was conducted to evaluate how many single nucleotidepolymorphisms (SNPs) are unique HJA-114-1011 on chromosome 9. Thesequence capture data of twenty-five elite inbred jalapeno lines and 219lines from other germplasm types was analyzed against HJA-114-1011. Twolines in the jalapeno group were derived from HJA-114-1011 and weregrouped with HJA-114-1011 for this analysis. The analysis consisted ofcalculating the allele frequency for each SNP within the two groups:HJA-114-1011 derived, and non-derived jalapenos. When looking across thechromosome, there is a significant increase of number of unique SNPs toHJA-114-1011 in the QTL region. In total, 41 SNPs were found to beunique to HJA-114-1011 in a 560 kb region co-localized to the QTLregion. Methods developed to detect these unique SNPs can be used totrack the HJA-114-1011 nematode resistance trait within all types ofpepper germplasm.

1.13. (canceled)
 14. A method for producing a Capsicum annuum plantexhibiting resistance to root knot nematodes, comprising: a) crossing aCapsicum annuum plant comprising an introgressed allele on chromosome 9that confers increased resistance to root knot nematodes compared to aplant not comprising said allele, wherein said plant is a non-jalapenovariety, and wherein said introgressed allele is located betweenMarker_6 (SEQ ID NO: 1) and Marker_10 (SEQ ID NO: 21) on chromosome 9with itself or with a second Capsicum annuum plant of a differentgenotype to produce one or more progeny plants; and b) selecting aprogeny plant comprising said introgressed allele.
 15. The method ofclaim 14, wherein selecting said progeny plant comprises identifying agenetic marker genetically linked to said introgressed allele.
 16. Themethod of claim 15, wherein selecting said progeny plant comprisesidentifying a genetic marker within or genetically linked to a genomicregion between 250,338,645 bp and 250,504,749 bp on chromosome 9 on thephysical map of CM334 v1.55.
 17. The method of claim 16, whereinselecting said progeny plant comprises identifying a genetic markerwithin or genetically linked to a genomic region flanked in the genomeof said plant by marker locus Marker_7 (SEQ ID NO: 6) and marker locusMarker_9 (SEQ ID NO: 16).
 18. The method of claim 17, wherein selectinga progeny plant comprises detecting at least one polymorphism at a locusselected from the group consisting of marker locus Marker_7 (SEQ ID NO:6), marker locus Marker_8 (SEQ ID NO: 11), and marker locus Marker_9(SEQ ID NO: 16).
 19. The method of claim 14, wherein said progeny plantis an F₂-F₆ progeny plant.
 20. The method of claim 14, wherein producingsaid progeny plant comprises backcrossing.
 21. The method of claim 20,wherein backcrossing comprises from 2-7 generations of backcrossing. 22.A method of producing a Capsicum annuum plant exhibiting resistance toroot knot nematodes, comprising introgres sing into a plant a root knotnematode resistance allele, wherein said resistance allele is defined aslocated in a genomic region between 250,338,645 bp and 250,504,749 bp onchromosome 9 on the physical map of CM334 v1.55.
 23. The method of claim22, wherein said genomic region is flanked by Marker _7 (SEQ ID NO: 6)and marker locus Marker _9 (SEQ ID NO: 16).
 24. The method of claim 22,wherein said introgressing comprises backcrossing.
 25. The method ofclaim 22, wherein said introgres sing comprises marker-assistedselection.
 26. The method of claim 22, wherein said introgressingcomprises assaying for said root knot nematode resistance. 27.-38.(canceled)
 39. A method of selecting a Capsicum annuum plant exhibitingresistance to root knot nematodes, comprising: a) crossing a Capsicumannuum plant comprising an introgressed allele on chromosome 9 thatconfers increased resistance to root knot nematodes compared to a plantnot comprising said allele, wherein said plant is a non-jalapenovariety, and wherein said introgressed allele is located betweenMarker_6 (SEQ ID NO: 1) and Marker_10 (SEQ ID NO: 21) on chromosome 9with itself or with a second Capsicum annuum plant of a differentgenotype to produce one or more progeny plants; and b) selecting aprogeny plant comprising said introgressed allele.
 40. The method ofclaim 39, wherein selecting said progeny plant comprises identifying agenetic marker genetically linked to said introgressed allele.
 41. Themethod of claim 39, wherein selecting said progeny plant comprisesidentifying a genetic marker within or genetically linked to a genomicregion between 250,338,645 bp and 250,504,749 bp on chromosome 9 on thephysical map of CM334 v1.55.
 42. The method of claim 39, whereinselecting said progeny plant comprises identifying a genetic markerwithin or genetically linked to a genomic region flanked in the genomeof said plant by marker locus Marker_7 (SEQ ID NO: 6) and marker locusMarker_9 (SEQ ID NO: 16).
 43. The method of claim 41, wherein selectinga progeny plant comprises detecting at least one polymorphism at a locusselected from the group consisting of marker locus Marker_7 (SEQ ID NO:6), marker locus Marker_8 (SEQ ID NO: 11), and marker locus Marker_9(SEQ ID NO: 16).
 44. The method of claim 39, wherein said progeny plantis an F₂-F₆ progeny plant.
 45. The method of claim 39, wherein producingsaid progeny plant comprises backcrossing.
 46. The method of claim 45,wherein backcrossing comprises from 2-7 generations of backcrossing.47.-51. (canceled)